European Genome-Phenome Archive

File Quality

File InformationEGAF00000659975

File Data

Base Coverage Distribution

This chart represents the base coverage distribution along the reference file. Y-axis represents the number of times a position in the reference file is covered. The x-axis represents the range of the values for the coverage.

Data is represented in a log scale to minimise the variability. A high peak in the beginning (low coverage) and a curve descending is expected.

68 819 34335 517 63913 520 4676 620 3732 662 3311 796 875924 214706 001446 678354 194256 501206 109161 064134 819110 71294 58781 04371 50961 84855 71849 78344 55740 79837 10333 72231 75229 66827 47225 47624 17922 70021 60020 54018 80918 35217 75017 09016 66415 47515 21214 56014 25314 09513 89313 01112 89512 75612 29412 23311 77011 84711 41611 27310 85810 53210 64910 21510 19010 0799 6639 7329 4289 4229 0308 8878 7908 8118 6588 7688 6728 5228 3137 9628 1577 8357 9208 0017 8427 5697 5127 5657 3017 4287 1307 0646 6616 8346 5896 6376 6966 7716 5176 8316 5436 4716 5726 3736 3966 3666 4146 1936 0996 0846 0616 0486 0416 0525 8305 8925 8695 7365 7485 5995 7525 6515 5625 4165 4015 3755 5115 2475 4525 1965 2865 1985 1805 1034 9535 0315 1575 0445 1245 1044 9234 9034 9355 0005 1165 1364 9624 9195 0384 9674 8864 8944 7694 8054 6564 6794 6074 6384 5754 6534 4244 5284 5854 4834 4924 3854 4804 4694 5154 3874 3814 4624 4574 3754 3374 2974 3454 3564 3694 2884 2914 2534 3584 3444 4384 2904 2934 2594 1324 2284 2164 0994 1284 1294 0314 0273 9694 0233 9544 1034 1133 8183 8773 9753 8313 9823 9964 0873 7933 7413 7553 8083 8343 8723 8393 9493 7483 8343 7233 7793 7513 7043 7383 6593 6643 6533 6383 4623 5093 4723 5293 3913 3333 6313 4923 3333 3633 3923 4703 3143 2333 3593 2913 3513 2933 3813 2383 2583 2763 2213 2793 2353 1573 1573 0703 1213 1393 0703 0503 1963 1323 1363 0882 9723 0292 9783 0772 9272 9752 9512 8863 0612 8742 9102 8572 9692 7582 8302 7052 7192 7412 7172 5952 6832 6402 6912 6562 6352 5562 6622 4662 4702 5592 5172 5022 3992 4902 4242 3302 4312 4252 3512 2592 3322 2632 2662 2592 2482 1732 2052 1942 1192 2442 1172 0652 0782 1092 0941 9881 9892 0291 9581 9521 9241 9641 8291 7941 9211 9411 8891 8381 9181 8381 8761 9041 8341 9021 8911 8061 8871 8981 8371 7471 8511 7731 7461 7461 6961 6571 6391 6761 6661 6301 6731 5891 6861 5641 5721 5351 5281 5661 4281 5521 5501 4781 4921 4841 4791 5361 5011 4351 5101 4341 3691 3531 3301 3841 3191 2441 3401 3491 3371 3101 2831 3061 2631 2941 3401 3021 2631 2751 2341 1911 2741 2781 1971 1861 2411 1641 2371 1861 1471 1781 2311 1371 1131 1581 1251 1191 0841 1151 0419921 0681 0671 0421 00695798299693398693993694793791490294388288190292281485791988090684582483983278777280380775083780879979580378674274277271271771072969973973669669368366765962263064166562069959059862058355056457757456154053153652951451550649353848552051150050245046645950347750043747148948645342848244245843145544141342238241436940241138638840341039438235337634234735033935336732431830728530030732229726229926927126929527026927324623324721821220324821023722223320524023422125522123521321123723120020221718518818818317620918717417717016918316615518316418418416516717415715218015716817214315517416216116415517615016615813914713215115216113815714213413013913613015411212113410413812412210612210612511696121112114110101107901039095909279798375715977676173727666626966668161526768667670695462576364616064587758637249544442585937393641453942314447363727313836333026403525293133223531402629381821174025203431362732373032382933322726262226222424332819273220261930222824262824291722233027233320242424252324212221182828192123212319222822202321231912201815163123122219161719141913161710172015191120231120132117161416121812191718161314168101918171716131213111881613101314968141511128761512917181210151315151281110918181913201315171410111314141714131014169101010891715117121715127131119122122191625181216111214141891611129131511816410871268117106766368688718139788128312129688269710347931092 702100200300400500600700800900>1000Coverage value101001k10k100k1M10M# Bases

Base Quality

The base quality distribution shows the Phred quality scores describing the probability that a nucleotide has been incorrectly assigned; e.g. an error in the sequencing. Specifically, Q=-log10(P), where Q is the Phred score and P is the probability the nucleotide is wrong. The larger the score, the more confident we are in the base call. Depending on the sequencing technology, we can expect to see different distributions, but we expect to see a distribution skewed towards larger (more confident) scores; typically around 40.

344 5460000729 335365 3162 030 7601 439 542474 644880 018352 394391 002639 792260 924869 550608 886726 7581 288 813676 9131 217 210717 4651 310 1291 854 9151 950 0272 209 1033 617 8843 529 8783 241 8493 660 0827 950 15713 281 9617 919 61014 412 22429 243 02141 225 08220 694 81857 682 18339 138 53969 268 30381 544 343170 893 87400510152025303540Phred quality score0M20M40M60M80M100M120M140M160M# Bases

Mapped Reads

Number of reads successfully mapped (singletons & both mates) to the reference genome in the sample. Genetic variation, in particular structural variants, ensure that every sequenced sample is genetically different from the reference genome it was aligned to. Small differences against the reference are accepted, but, for more significant variation, the read can fail to be placed. Therefore, it is not expected that the mapped reads rate will hit 100%, but it is supposed to be high (usually >90%). Calculations are made taking into account the proportion of mapped reads against the total number of reads (mapped/mapped+unmapped).

99.1 %7 774 02999.1 %0.9 %

Both Mates Mapped

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. This chart shows the fraction of reads in pairs where both of the mates successfully map to the reference genome. .

Notice that reads not mapped to the expected distance are also included as occurs with the proper pairs chart.

98.8 %7 751 47498.8 %1.2 %

Singletons

When working with paired-end sequencing, each DNA fragment is sequenced from both ends, creating two mates for each pair. If one mate in the pair successfully maps to the reference genome, but the other is unmapped, the mapped mate is a singleton. One way in which a singleton could occur would be if the sample has a large insertion compared with the reference genome; one mate can fall in sequence flanking the insertion and will be mapped, but the other falls in the inserted sequence and so cannot map to the reference genome. There are unlikely to many such structural variants in the sample, or sequencing errors that would cause a read not to be able to map. Consequently, the singleton rate is expected to be very low (<1%).

0.3 %22 5550.3 %99.7 %

Forward Strand

Fraction of reads mapped to the forward DNA strand. The general expectation is that the DNA library preparation step will generate DNA from the forward and reverse strands in equal amounts so after mapping the reads to the reference genome, approximately 50% of them will consequently map to the forward strand. Deviations from the 50%, may be due to problems with the library preparation step.

50 %3 924 27950 %50 %

Proper Pairs

A fragment consisting of two mates is called a proper pair if both mates map to the reference genome at the expected distance according to the reference genome. In particular, if the DNA library consists of fragments ~500 base pairs in length, and 100 base pair reads are sequenced from either end, the expectation would be that the two reads map to the reference genome separated by ~300 base pairs. If the sequenced sample contains large structural variants, e.g. a large insertion, where we expect the reads mapping with a large separation would be a signal for this variant, and the reads would not be considered as proper pairs. Based on the sequencing technology, there is also an expectation of the orientation of each read in the fragment.

The rate of proper pairs is expected to be well over 90%; even if the mapping rate itself is low as a result of bacterial contamination, for example.

98.4 %7 722 49498.4 %1.6 %

Duplicates

PCR duplicates are two (or more) reads that originate from the same DNA fragment. When sequencing data is analyzed, it is assumed that each observation (i.e. each read) is independent; an assumption that fails in the presence of duplicate reads. Typically, algorithms look for reads that map to the same genomic coordinate, and whose mates also map to identical genomic coordinates. It is important to note that as the sequencing depth increases, more reads are sampled from the DNA library, and consequently it is increasingly likely that duplicate reads will be sampled. As a result, the true duplicate rate is not independent of the depth, and they should both be considered when looking at the duplicate rate. Additionally, as the sequencing depth in increases, it is also increasingly likely that reads will map to the same location and be marked as duplicates, even when they are not. As such, as the sequencing depth approaches and surpasses the read length, the duplicate rate starts to become less indicative of problems.

32.9 %2 581 68032.9 %67.1 %

Mapping Quality Distribution

The mapping quality distribution shows the Phred quality scores describing the probability that a read does not map to the location that it has been assigned to (specifically, Q=-log10(P), where Q is the Phred score and P is the probability the read is in the wrong location). So the larger the score, the higher the quality of the mapping. Some scores have a specific meaning, e.g. a score of 0 means that the read could map equally to multiple places in the reference genome. The majority of reads should be well mapped, and so we expect to see this distribution heavily skewed to a significant value (typically around 60). It is not unusual to see some scores around zero. Reads originating from repetitive elements in the genome will plausibly map to multiple locations.

326 7581 1295862 0479221 2239681 6611 6858 8386 4992 87011 7423 1262 00931 2483 39313 24710 6863 38318 23869116 03458 3225374 046440412545271 5861 1198468981 1628941 47435 178130 1644 0541 3944 8044 1469988 3321 9902 69430 3344 5344 5366 0309 8644 61212 6049 86813 87424 69445 7586 676 832051015202530354045505560Phred quality score0.5M1M1.5M2M2.5M3M3.5M4M4.5M5M5.5M6M6.5M# Reads

Mapped vs Unmapped

Stacked column chart for both mapped and unmapped reads along all chromosomes in the reference file. It is a similar representation as shown in the Mapped reads chart but for each chromosome. Although sequenced sample may be a female, it is possible to get reads in the Y chromosome as there are common regions in both chromosomes called pseudoautosomal regions (PAR1, PAR2).

Unmapped reads belonging to each chromosome are determined when the one mate/pair is aligned and the other is not. The unmapped read should have chromosome and POS identical to its mate. It could also be due when aligning is performed with bwa as it concatenates all the reference sequences together, so if a read hangs off of one reference onto another, it will be given the right chromosome and position, but it also be classified as unmapped.

100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%100%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%0%123456789101112131415161718192021XYM0%10%20%30%40%50%60%70%80%90%100%mappedunmapped